Microalgae are simple aquatic organisms that produce oxygen and organic matter by photosynthesis. Microalgae have uses in the production of food, nutritional supplements, pharmaceuticals, natural pigments, biochemicals, and biomass for fuel production. They have also utility in the removal of nitrogen, phosphorus and heavy metals in waste water. Microalgae are particularly useful because of their high growth rate and tolerance to various environmental conditions.
Due to the wide range of uses of microalgae and microalgae-based products, effective methods for growing and harvesting microalgae are essential. “Conditioning” is the treatment of an aqueous microalgae dispersion by cell rupture, chemical treatment, and/or flocculation in order to facilitate isolation of the microalgae or cellular components in a subsequent step.
Two basic approaches exist for the culture of microalgae: closed bioreactor systems and open pond systems. The most capital intensive system is the closed bioreactor which utilizes transparent conduits in which the microalgae grow in water by exposure to light and introduction of carbon dioxide and nutrients. Major reasons for choosing the bioreactor design are control of the culture and/or the desire to remove carbon dioxide from waste gas emissions. With a genetically-modified microalgal species, isolation helps to prevent contamination by other species and escape into the environment.
Harvested microalgae biomass can be converted to animal feed, solid fuel, methane, hydrogen, synthesis gas, or liquid transportation fuels such as biodiesel, and bioethanol. Sequential operations may allow production of two or more of these products from the microalgal lipids (triglycerides), starches and residues. Of special interest herein is the production of fatty acid esters (biodiesel) from the microalgal lipids. See, for example, the research review paper: “Biodiesel from microalgae,” Yusuf Chisti, Biotechnology Advances, volume 25, pages 294-306 (2007), the contents of which are incorporated herein by this reference. Harvested microalgae biomass may also be hydrotreated for the production of liquid hydrocarbons.
The optimum population density for microalgae cultivation is one where light reaches the full depth of the growing medium, without upper layers substantially shading the lower. The range of 200,000 to 500,000 cells per milliliter is commonly used within the art as an efficient population density for growth of unicellular microalgae. In agitated ponds and bioreactors, higher populations can be maintained, while in static ponds, lower densities perform best. These population densities result in low concentrations (on the order of 0.02 weight percent) of microalgal products in the culture medium.
Pre-concentration of microalgae before harvesting is a desirable step in order to reduce the volume of microalgae culture handled. However, pre-concentration is difficult and expensive to implement in large scale aquaculture systems such as would be needed for microalgal biofuel production. Except in the production of very high-value microalgal products, current microalgae harvesting technology is uneconomical for handling the large volume of growth media. In order for microalgae to become an economical, renewable source of low-value, high-volume products (such as biofuels), improved methods for growing, harvesting, and concentrating microalgae are needed.
Numerous techniques have been tried for removing microalgae from a liquid stream. Filtration is the most common process for isolating solids from liquid dispersions and many configurations of filters have been used or attempted with microalgae. However many useful species of microalgae are not amenable to filtration due to their very small size and/or their soft, deformable structure which causes plugging of the filter. U.S. Pat. No. 5,490,924, the contents of which are incorporated herein by this reference, describes a filter system having a backwash and filter cleaning system adapted to simultaneously vertically-reciprocate and rotate to selectively dislodge microalgae and other particulates from the filter. This system is suitable for water purification, but not for large-scale microalgae biomass isolation. U.S. Pat. No. 3,875,052, the contents of which are incorporated herein by this reference, details a multi-step process which comprises a pre-concentration step in which the microalgae suspension is fed along a filtration surface at a high contact velocity, followed with filtration, washing and pressing. However this process is only amenable to filamentous microalgae and not to smaller, single-celled microalgae.
U.S. Pat. No. 6,328,165, the contents of which are incorporated herein by this reference, describes a harvesting apparatus for marine aquaculture which uses a complex moving belt filter and incorporates wash cycles.
U.S. Pat. No. 3,951,805, the contents of which are incorporated herein by this reference, is a complex and expensive belt filter device for harvesting microalgae. There are many more filtering techniques that can be utilized, but in all of them, particular attention must be paid to the clearing or cleaning of the filter to insure efficient operation. If the microalgae can deform, it can quickly plug or blind the filter.
Centrifugation is another common solids isolation technique which is sometimes cited for use in microalgae isolation. For example, U.S. Pat. No. 4,115,949, the contents of which are incorporated herein by this reference, describes the culture and centrifugation of microalgae for the production of glycerol and proteinaceous substances of nutritive value. Centrifugation, filtration, and sedimentation are methods of microalgae harvesting that were discussed in “A Look Back at the U.S. Department of Energy's Aquatic Species Program: Biodiesel from Algae,” NREL/TP-580-24190 (1998), the contents of which are incorporated herein by this reference. Unfortunately centrifugation equipment is too expensive for the initial step of large-scale microalgae harvesting for low-cost, high-volume products.
U.S. Pat. No. 6,332,980, the contents of which are incorporated herein by this reference, describes the use of dissolved air flotation and a hydrocyclone separator to remove volatile gases, pesticides, and particles such as microalgae from water. This system is suitable for water purification but not for large-scale microalgae biomass isolation.
Acoustic energy has also been used to separate particles, including microalgae, from the carrier liquid. U.S. Pat. Nos. 4,055,491 and 5,626,767, the contents of each of which are incorporated herein by this reference, describe the use of an ultrasonic resonance wave to move particles, including microalgae, at different rates, allowing them to be separated. However, this technique relies on differences in the acoustic properties between the solid and the liquid, and since microalgae are occasionally neutrally buoyant, a large amount of energy would be required for the separation.
Harvesting microalgae on an adsorbent having a hydrophobic surface was disclosed by Curtain, et al. in U.S. Pat. No. 4,554,390, the contents of which are incorporated herein by this reference. This process is practical in the production of high value products but it is too expensive for biofuel applications.
Adsorptive bubble separations are a group of processes used in treating a feed dispersion that comprises a carrier liquid and hydrophobic material that is molecular, colloidal, and/or particulate in nature. This hydrophobic material is selectively collected (i.e., adsorbed or attached) to the surface of bubbles such that they can be allowed to rise through the carrier liquid, thereby concentrating or separating the hydrophobic material from the carrier liquid. The resulting froth with collected particles may be treated in one of several ways to collapse the froth and isolate the particles. This important process is commercially utilized in a wide range of applications that include isolation of minerals and metals from ore, dewatering of microalgae, removal of oil droplets from an aqueous stream, removal of ash particulates from coal, removal of particles in waste-water treatment streams, purification of drinking water, and removal of ink and adhesives during paper recycling. See for example, “Harvesting of Algae by Froth Flotation,” G. V. Levin, et al., Applied and Environmental Microbiology, volume 10, pages 169-175 (1962), the contents of which are incorporated herein by this reference. Other applications of adsorptive bubble processes are described in Adsorptive Bubble Separation Techniques, Robert Lemlich, Editor, Academic Press, New York, N.Y. (1972) which is hereby incorporated by reference. In all of these applications, there is a need to efficiently contact particles or droplets in an aqueous dispersion with a gas and then attach the hydrophobic material to the bubbles. This process is amenable to very large feed streams and is widely practiced as such in the mining industry.
For adsorptive bubble processes to separate materials, hydrophobic materials comprise the component to be separated. Rendering material hydrophobic is commonly called “conditioning”, wherein particle surfaces are treated with chemicals, or other techniques that selectively modify the component to be separated. In most cases, the particles are not initially hydrophobic, and the particles to be separated or dewatered are made hydrophobic so they may be collected and separated with an adsorptive bubble process. In other cases, the particles are all hydrophobic, and one component is modified to make it hydrophilic in order to keep it in the aqueous stream.
Microalgae are hydrophilic therefore adsorptive bubble separation is minimally effective on whole, live microalgae cells. In order to use adsorptive bubble separation, microalgae cells must be conditioned to make them hydrophobic.
An example of the use of flocculation conditioning followed by adsorptive bubble separation to harvest microalgae was disclosed in U.S. Pat. No. 4,680,314, the contents of which are incorporated herein by this reference. U.S. Pat. No. 6,524,486, the contents of which are incorporated herein by this reference, also utilizes a flocculating agent to cause accumulations of microalgae that are then floated out using an adsorptive bubble process. This process requires the addition of flocculating agents, which are expensive, may have environmental concerns, and can contaminate the product or the growth medium.
Another method to render the microalgae cells hydrophobic so as to use adsorptive bubble separation is by “cell rupture” (also referred to as “cell disruption” or “lysis”). By rupturing the cell wall and/or cell membrane, lipids and other naturally hydrophobic components are released from the cell. These components and cell fragments can then be recovered with an adsorptive bubble separation process.
Cell rupture can be achieved by a number of methods which can be classified as chemical, physical or mechanical. The chemical methods include enzymatic digestion, detergent solubilization, lipid dissolution with a solvent, and alkali treatment (lipid saponification). Physical methods include osmotic shock, decompression, sonication, heat treatment, and freeze-thawing. Mechanical methods include grinding, high shear homogenization and pressure extrusion. High speed impeller homogenization of cells (e.g., kitchen blender) is a mechanically simple process, but requires high energy and heat removal. The large energy requirement per volume of microalgal media renders this technique uneconomical for large-scale aquaculture systems. See for example, U.S. Pat. No. 4,931,291, the contents of which are incorporated herein by this reference, which uses various microalgal cell rupture methods to produce feed for crustacean and shellfish larvae.
A common cell disruption process in the prior art uses a pump to force the feed mixture at high pressure through a restricted orifice valve. Cell disruption is accomplished by three different mechanisms: impingement on the valve, high liquid shear in the orifice, and sudden pressure drop upon discharge, causing finally an explosion of the cell. As an example of this, the MICROFLUIDIZER™ cell disruption equipment of Microfluidics, Newton, Mass., US utilizes pressures of about 5,000 to 40,000 prig (345-2760 bar). U.S. Pat. No. 6,405,948, the contents of which are incorporated herein by this reference, describes a method for liberating intracellular materials using a resonance disintegration mill in which a high speed rotor creates a series of compressions and decompressions.
U.S. Pat. Nos. 5,776,349 and 6,000,551, the contents of which are incorporated herein by this reference, disclose that microalgae cells are ruptured when the microalgal feed dispersion is subjected to a pressure drop created by pumping through an orifice. Pressure drops of 50 to 200 psig (3.4-14 bar) are claimed to render an acceptable percentage of the cells recoverable with an adsorptive bubble separation. However, it is expensive to pump the entire feed dispersion, where the growing media may represent greater than 99% of the mass, at these pressures to obtain a high percentage of cell rupture.
Fine grinding technology has application to microorganism cell disruption at the laboratory scale. In ball or bead mills, cells are agitated in suspension with grinding media which are small abrasive particles such as glass or ceramic beads. Cells break because of shear forces, grinding between beads, and collisions with the beads. U.S. Pat. No. 5,330,913, the contents of which are incorporated herein by this reference, claims that an aqueous suspension of Chlorella cells is disrupted by the rapid pressure changes created by a rotating impeller within a small, cylindrical, tight-sealed container with rigid spheres having a constant diameter of 500 to 800 microns.
Horizontal or vertical, high-energy, disk mills employ a chamber containing disks on a high speed rotor and grinding media. The grinding process may be done batch-wise or continuously. This design is used for grinding pigments, dyes, pharmaceuticals, food products, minerals, and small quantities of biological cells.
U.S. Pat. No. 6,589,785, the contents of which are incorporated herein by this reference, describes a method of disrupting cells by freezing them into solids and fracturing them with a vibratory ball mill in the presence of denaturing substances. In the example cited, a device called the DISMEMBRATOR U was used to fracture small amounts of the cells. Any process requiring freezing the feed would be prohibitively expensive.
U.S. Pat. No. 5,374,522, the contents of which are incorporated herein by this reference, claims a method for rupturing microorganisms by the use of ultrasonic energy in the presence of small beads. U.S. Patent Application 2006/0084165, the contents of which are incorporated herein by this reference, describes a method of disrupting cells or viruses. The method involves adding magnetic beads to a solution containing cells or viruses, vibrating the magnetic beads and irradiating a laser upon the magnetic beads to disrupt the cells. Once again, these techniques are difficult and expensive to implement in large scale aquaculture systems such as would be needed for algal biofuel production.